RIP assays were performed essentially as previously described [14 (link)]. In brief, QGY-7703 or MHCC-97H cells were harvested and lysed (5 mM HEPES [pH 7.4], 85 mM KCl, 0.5% NP40, 1 mM DTT, 5 mM PMSF, supplemented with protease inhibitors cocktail (Roche, Switzerland), RNase inhibitors (Invitrogen, USA) for 8 min on ice. After centrifugation, the supernatant was collected and sonicated and 10% of the lysate serves as ‘input’. The remainder of the lysate was incubated with 40 μl protein G-coupled dynabeads (Life Technologies, USA) for 30 min at 4 °C to decrease the background, followed by washing in lysis buffer and adding protein G-coupled dynabeads with 3 μg anti-STAT3 antibody, anti-flag antibody or IgG control, then rotated overnight at 4 °C. RNA was isolated by TRIzol (Invitrogen, USA), incubated with DNase I (Sigma, USA) and reverse-transcribed into cDNA, and subjected to qRT-PCR detection (primers in Additional file 1 : Table S2).
Protein g coupled dynabeads
Manufactured by Thermo Fisher Scientific
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Protein G–coupled Dynabeads are magnetic beads designed for the purification and isolation of proteins and protein complexes. They provide a convenient and efficient method for capturing target proteins from complex biological samples.
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Lab products found in correlation
Thiolutin, by Abcam (2 mentions)
β actin gtx109639, by GeneTex (2 mentions)
5 foa, by Cayman Chemical (2 mentions)
Secondary antibodies against rabbit 7074s and mouse 7076s igg, by Cell Signaling Technology (2 mentions)
Iaid tag m214 3, by MBL Life Science (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Sv5 pk1, by Bio-Rad (2 mentions)
Mouse monoclonal anti myc 9e10, by Takara Bio (2 mentions)
Streptavidin coupled magnetic beads, by Thermo Fisher Scientific (2 mentions)
Phusion pcr master mix, by New England Biolabs (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Reducing agent, by Thermo Fisher Scientific (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Dynabeads, by Merck Group (1 mentions)
Flag antibody, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
21 protocols using protein g coupled dynabeads
1
RIP Assay for STAT3 Binding
2
Affinity Purification of RNA-Binding Proteins
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PAB1-TAP and GAPDH-ProtA strains were cross-linked as described and RBPs were purified by 2C. Non-irradiated samples from each strain were used as negative controls. Protein G–coupled Dynabeads (#10004D; Thermo Fisher Scientific) were incubated with rabbit IgG (#I5006; Sigma-Aldrich) for 30 min at room temperature. Unbound IgG was eliminated by washing the beads three times with buffer B, and fractions from the 2C eluates equivalent to 100 μg of RNA were incubated with the beads with gentle rotation at 4°C for 2 h. Unbound material was removed, and the beads were washed once with buffer B, three times with buffer C (25 mM Tris–HCl, pH 7.5; 1 M NaCl; 1.8 mM MgCl2; 0.5 mM DTT; and 0.01% NP-40), and another time with buffer B. Pab1-TAP was eluted by incubating the beads in buffer D (25 mM Tris–HCl, pH 7.5; 140 mM NaCl; 1.8 mM MgCl2; 0.5 mM DTT; and 0.01% NP-40) with 2.5 μg of TEV protease at 34°C for 1 h and GAPDH-ProtA was eluted by resuspending the beads in buffer D plus loading buffer and incubating them at 95°C for 5 min.
3
Affinity Purification of Phosphorylated Proteins
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Cells were grown to 95% confluency prior to lysis. Tyrosine phosphatase inhibition was achieved by addition of 100 μM sodium orthovanadate (Sigma-Aldrich) pre-activated with 30% H2O2 (1: 200) to cell medium for 20 minutes. Cells were washed with ice cold PBS before lysis. Proteins were extracted by scraping the cells in ice-cold lysis buffer, as described elsewhere45 , and normalized according to their protein concentrations as measured by Lowry. Lysates were incubated for 1h30 at 4 °C with M2 affinity resin (Sigma-Aldrich) for FLAG APs. For endogenous APs, lysates were incubated with 5 μg of antibody overnight at 4 °C. Protein G-coupled Dynabeads® (Thermo) were then added and incubated for 30 minutes at 4 °C. Beads were washed three times with lysis buffer. For Western blotting, beads were resuspended in 4x Laemmli buffer. For MS experiments, beads were additionally washed twice with 20 mM Tris pH 7.4 and proteins were eluted by incubating with agitation at 4 °C with 50 mM H3PO4 before digestion.
4
Affinity Purification of RNA-Binding Proteins
5
Genotypes and Reagents for Yeast Study
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Genotypes of strains and yeast plasmids used in this work are listed in Table S3 and S4 . All chemicals used in this study were purchased analytical grade from either Sigma-Aldrich or Carl Roth except for the following: Drop Out Mix for yeast synthetic medium (SD) was from US Biological Life Sciences (D9543-01); 5-FOA was bought from Cayman Chemical (17318); Ampure XP beads were from Beckman Coulter (A63881); Protein-G coupled dynabeads from Thermo Fisher Scientific (10009D) and Thiolutin from Abcam (ab143556). Secondary antibodies against rabbit (7074S) and mouse (7076S) IgG coupled to horseradish peroxidase (HRP) were purchased from Cell Signaling. Antibodies against H3K56ac were from Active Motif (39281), G6PDH (A9521) and Myc (MABE282) from Sigma, iAID-tag (M214-3) from MBL International and β-actin (GTX109639) from GeneTex. Antibody dilutions were as suggested by the manufacturer (Table S12 ).
6
UV-Induced β-catenin Immunoprecipitation
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Untransfected MCF7 or MDA-MB-231 cells (5×106) in an open dish on ice were placed in a Stratalinker UV-light box and irradiated at 254 nm for 2 min. DMEM was removed and 1 ml cold PBS added to each plate. Cells were scraped, collected into tubes and centrifuged at 100 × g for 5 min at 4°C. PBS was removed and cells were lysed in cell lysis buffer (5 mM HEPES pH 7.4, 85 mM KCl, 0.5% NP40) for 8 min at 4°C with shaking. Samples were transferred to tubes and centrifuged at 2,500 × g for 5 min at 4°C. Supernatant lysate was precleared by incubation with 40 µl protein G-coupled Dynabeads (Thermo Fisher Scientific, Inc.) for 30 min at 4°C. Following centrifugation (2,500 × g, 5 min, 4°C), the supernatant was collected and protein G-coupled Dynabeads were discarded. Then 40 µl protein G-coupled Dynabeads and 3 µg anti-β-catenin antibody (1:1,000; cat. no. 8480; CST Biological Reagents Co., Ltd.) were added to the supernatant and mixed overnight at 4°C. Following, beads were collected by centrifugation at 2,500 × g for 5 min at 4°C and washed 4× with lysis buffer (5 mM HEPES pH 7.4, 85 mM KCl, 0.5% NP40). RNA was isolated from the beads using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), incubated with DNase I (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
7
BNIP3/BAX Immunoprecipitation and Analysis
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Immunoprecipitation were performed using protein G-coupled dynabeads (ThermoFisher Scientific) [36 (link)]. Lysed proteins (500 µg) were incubated with 2 µg BNIP3/BAX antibody over night at 4 °C with shaking in a PBS buffer containing 1 mM DTT, 0.005% Brij35 and protease-phosphatase inhibitors. After 24 h, 20 µL dynabeads were added and the solution was incubated again for 1 h. The precipitated immune complex was washed twice and then resuspended in an elution buffer containing lithium dodecyl sulfate (LDS)-Sample Buffer (1:4) and Reducing Agent (1:10) (ThermoFisher Scientific) in PBS and heated for 5 min at 95 °C. After removal of the dynabeads, the eluate was analyzed via immunoblotting.
8
Immunoprecipitation of FLAG-tagged Proteins
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Whole cell protein extract from 45 × 106 mESCs was obtained by lysis in 500 µl Buffer B (final: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 10% Glycerol, 1 mM DTT, 1 mM PMSF, 0.2% NP-40, 1× Complete Mini protease inhibitor). Lysate was homogenized by sonication in a Diagenode Bioruptor followed by rotation for 3 h at 4 °C. After 30 min centrifugation at 4 °C protein concentration of the lysate was determined by Bradford assay (Biorad). In parallel, 30 µl Protein G coupled Dynabeads (Thermo Fisher Scientific) were prepared for each IP reaction as follows: 3x wash in Buffer B, incubation with 1.5 µg FLAG antibody (Sigma Aldrich, F1804) for 3 h at 4 °C, 1× wash in Buffer B and finally resuspension in 30 µl of Buffer B. For each IP, 30 µl of pre-bound Dynabeads were incubated with 3 mg protein extract in a final volume of 500 µl overnight at 4 °C. Finally, beads were washed four times with Buffer B and proteins were eluted at 95 °C in SDS sample buffer and analyzed by Western blots.
9
Co-Immunoprecipitation of Protein Complexes
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Co-IP was performed with Protein G-coupled Dynabeads® (Thermo Fisher, Waltham, MA, USA) according to manufacturer’s instructions. Briefly, 30 μl beads were incubated with 3 μg anti-Flag antibody (F1804; Sigma-Aldrich) for 10 min in PBS-T (0.2% Tween-20) at room temperature. Antibody-coupled beads were incubated overnight with 200 µg protein lysate in PBS-T at 4 °C. After washing beads in PBS-T, protein complexes were eluted in SDS loading buffer for 5 min at 70 °C. Subsequently SDS-PAGE with pre-cast 4–20% gradient gels (Bio-Rad, Feldkirchen, Germany) and Western Blotting were performed as previously described [33 (link)]. Proteins were detected with primary antibodies against Flag (F1804; Sigma-Aldrich), MYSM1 (orb137033, Biorbyt; St Louis, MO, USA), H2Aub (8240; Cell Signaling, Leiden, The Netherlands), H2AX (Abcam; ab2893, Cambridge, UK), Actin (sc-1615HRP; Santa Cruz, Heidelberg, Germany), and other specific antibodies as indicated followed by Peroxidase-coupled specifies-specific secondary antibodies (711-035-152, 715-035-150; Jackson ImmunoResearch, West Grove, PA, USA).
10
Immunoprecipitation and Western Blot Analysis
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Gene description Probes ID For the immunoprecipitation (IP) assay, 250 μg of protein (cell lysates) in 500 μL of RIPA buffer was incubated overnight at 4°C with unspecific mouse/rabbit/rat IgG antibodies (Jackson Immunoresearch) or rat anti-HA-tag (Roche Diagnostics) or mouse anti-FlagM2 and rabbit anti-APP-CTF (Sigma-Aldrich) at 4 ng/μL and then pulled-down for 2 hours using 50 μL of protein G-coupled dynabeads (Thermo Fisher Scientific). After washing three times with 0.3 M NaCl solution, samples were denatured and subjected to western blot (WB) using APP-CTF, anti-FlagM2, or anti-HA antibodies, and the corresponding horseradish peroxidase-conjugated secondary IgG antibodies (Jackson Immunoresearch). The EasyBlocker kit was used to limit unspecific signal according to the manufacturer's recommendations (GeneTex, Euromedex, Souffelweyersheim, France).
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